The present invention relates to a process for producing biologically active factors from a substrate in the form of cell homogenates of organ tissues, of micro-organisms, of plant material and/or of body liquids. More specifically, the invention is directed to obtaining sterile, biologically active factors which may be used for the stimulation and, on the other hand, for the inhibition of processes of growth, metabolism, and of synthesis in eukaryotic and prokaryotic cells and tissues.
On the basis of data from my own experiments with respect to effects on growth and metabolism of human tissue cells in cell cultures by treatment with macromolecular organ extracts (V. Paffenholz and K. Theurer: Einfluss von makromolekularen Organsubstanzen auf menschliche Zeller in vitro, I. Diploide Kulturen, II. Tumorzellkulturen (the effects of macromolecular organ substances on human cells in vitro, I. diploid cultures, II. tumor cell cultures): Der Kassenarzt, no. 27, pages 5218 to 5226, 1978, no. 19, pages 1876 to 1887, 1979), it has been seen than an inhibitory effect on tumor cells and heteroploid cells was produced at high dilutions in a concentration of 10.sup.-6 to 10.sup.-9 g/ml of culture medium, and stimulation of healthy normal cells was produced using high concentrations between 10.sup.-3 to 10.sup.-6 g/ml of culture medium. The active factors were identified as proteins and peptides.
In view of these differences, it seemed likely that different factors were effective for inhibition and stimulation with different sites of action (K. Theurer: Prophylaxe und Therapie von Praekanzerosen und Malignomen mit makromolekularen Organextrakten (prophylaxis and therapy of precanceroses and malignant tumors with macromolecular organ extracts): Krebsgeschehen 4, pages 80 to 86, 1980). This was responsible for the development of the present process for separating inhibiting and stimulating factors from cell homogenisates. Studies of S. R. Burzynsky, Z. Stolzmann, B. Szopa, E. Stolzmann and O. P. Kaltenberg (Antineoplaston A in Cancer Therapy: Physiological Chemistry and Physics, 9, pages 485 to 500 (1977)), make it clear that such tumor inhibiting factors are present in the urine of healthy individuals and may be produced therefrom. It is furthermore known that tumor inhibiting factors are present in the blood of healthy individuals.
One purpose of the invention is that of producing active factors from biological substrates selectively and freeing them of undesired accompanying substances so that they may be used for scientific and therapeutic purposes and in industry.
In the present invention the substrate, in an aqeuous form, and free or freed of accompanying particulate substances is selectively separated using a biological affinity sorbent, in which at least one nucleic acid, or at least one protein or peptide, is coupled with a carrier substance. Non-binding components are contained in the primary eluate and the active factors, having an affinity, are secondarily eluated. In this respect, the wording "primary eluate" is used in the sense of an eluate which is provided by simple rinsing of the sorbent, while on the other hand, for producing or obtaining the eluate with the binding active substances in it, special adjuvants are used having the effect of desorbing the active substances or factors from the affinity sorbent. In this respect not only the primary, but furthermore the secondary eluate may undergo further fractionation, in which respect, in the case of the secondary eluate, physico-chemical fractionating operations are preferred.
With the selective biological separating process of the invention for active substances or factors it becomes possible for biologically active substances or factors having given properties to be separated positively on a substrate made up of a great number of different materials. Separation of the substrate into components is preferably undertaken by affinity chromatography or by a combination of affinity chromatography and electrophoresis. The electrophoresis may be undertaken in the form of "Free-flow-Electrophoresis". The affinity chromatography makes possible selective separation into or of desired active factors, in which respect the nucleic acids or the proteins, which are coupled with the carrier substance for forming the affinity sorbent, are selected in a way dependent on the desired properties of the desired active factor or factors. The nucleic acids, or, in the other case, the proteins or peptides, used for producing the affinity sorbent, are, for their part, preferably isolated from a substrate of the sort noted at the start. More specifically, if the nucleic acids, or in the other case, the proteins or peptides for the affinity sorbent, are to be isolated from such a substrate, it is best for the substrate to be freed, before separating off these substances, of undesired or ineffective accompanying substances. The undesired or ineffective accompanying substances may be enzymatically degraded, whereafter the products of degradation may be removed by dialysis of fragments or by ultrafiltration. In this respect, as a general rule, products with a molecular weight under 600 will be removed, because it has turned out that the biologically interesting active factors have a higher molecular weight, and, more specially one of the order of 600 to 1,000,000. The enzymatic degradation is preferably undertaken positively in a way dependent on the sort of desired active substances. Carbohydrates and lipids may, generally speaking, be removed, while on the other hand, proteins or nucleic acids will only be removed if they are undesired as active substances. If only polypeptides or proteins are desired as the active substances, this generally being the case, the nucleic acids may be degraded as well. If, the other way round, the desired active substances are nucleic acids, for example for further use as affinity sorbents, it is then possible for the polypeptides or proteins to be degraded. Desoxyribonucleic acid will most often be the preferred nucleic acid because, from the biological point of view, it is more interesting. For special purposes, however, ribonucleic acid may be used.
Before separating, and more specifically, before the removal of any undesired accompanying substances, the substrate is best sterilized. If a material change in the biological substances is not of key importance, conventional ways of sterilizing, as for example chemical sterilization or sterilization with ionizing radiation may be utilized. More importantly, however, sterilization by condensing volatile substances with sterilizing properties onto the substrate, in the form of a powder, is to be preferred, as the preferred starting material is also in the form of a powder; the substances for this purpose which are more specially preferred are those in the case of which any excess may be removed by evaporation or sublimation. Sterilization is best undertaken in vacuo using pressure variations.
The affinity sorbent may be produced by coupling highly purified nucleic acid fractions or protein (or, in the other case) peptide fractions to inert carrier substance for affinity chromatography of the substrate. The discovery has been made that carrier-bound protein or peptide fractions have a specially high degree of avidity for stimulating acting substances and carrier-bound nucleic acid fractions have such an avidity for inhibitory active substances, for which reason they are preferred for use on these lines for producing the active substances in question in the secondary eluate. In this respect, it is furthermore important from which biological material the protein or peptide fractions on the one hand, and the nucleic acid fractions on the other hand, are produced, which are used for producing the affinity sorbent, as explained in more detail hereinafter. By making the right selection, it is, for this reason, possible to make certain that given active factors, present in the substrate and having given properties, may be positively separated out.
The invention is directed not only to the process for producing or obtaining the active factors, but is furthermore directed to the active factors per se, which may be obtained with the process of the invention, and furthermore to the use of such substances. Detailed working examples will be given herein of the invention. In this respect, it is to be noted that the substrate may be produced from any sort of biological material containing active factors. The most important of such starting materials seem to be body fluids such as blood, cerebrospinal fluid, amnion-exudates, transsudates, and fluids expressed from tissues and urine.
In a preferred form, the process of the invention may be divided into a number of preferred steps, which may be combined together in any suitable way, namely:
(a) non-destructively sterilizing the starting materials to eliminate any viral components, while, at the same time, increasing the quality of the hydrophilic properties, that is, the water solubility by partial lysis of the molecular sub-units and the addition of radicals of the acids and alkalis used,
(b) separating (first-stage) ineffective or otherwise undesired components of the substrate,
(c) coupling of highly purified desoxyribonucleic acid and/or RNA fractions, more specially, ribosomal RNA and, on the other hand, of protein and peptide fractions to activated inert carrier substance for forming the affinity sorbent for affinity chromatography of the substrate to be separated into its components,
(d) separating (second-stage) the substrate, produced in step (b), on the affinity sorbent, prepared in step (c), by sample inlet and elution steps,
(e) obtaining tumor-inhibiting factors by affinity chromatography of substrates produced from fetal and/or juvenile normal tissue and/or body liquids of healthy individuals on carrier-bound DNA,
(f) producing stimulating factors,
(g) producing organ-specific fractions, and
(h) producing fractions from micro-organisms and plant cells.
The intermediates of process steps (a) may be used per se. The sort of use and further processing of the products made in this process will be given in further detail hereinafter. On the other hand, steps (a) and (b) are not necessary for processing the substrate. A particularly interesting aspect of the invention is, however, the sort of sterilization and first-stage treatment of the substrate to be separated and electrophoretic elution of the proteins, adsorbed on the carrier-bound substances, by changing the voltage or current level and furthermore the use of special starting materials and their application. Scientific details and the sort of the application and further processing are in line with the present stage of development of the art.
A condition for therapeutic use of active substances from biological tissues in sterility with respect to viruses. Government prohibition of processing medicaments with ionizing radiation and of denaturizing by chemical processing makes a specially non-destructive sterilization step (a) of key importance. This process step is furthermore beneficial with respect to undertaking further process steps later because, at the same time, there are better hydrophilic properties and the partial cleavage into molecular sub-units is effected. The acidic or alkaline effect produced by addition of acids and alkalis may readily be neutralized by the right form of buffering so as to make certain of the best pH-value in the separating operation.
In the case of cell cultures and animal experiments a significantly better inhibitory effect on tumors and a stimulating effect in the case of normal tissue was produced by preparations obtained using process step (a) than is the case with preparations which only underwent freeze-drying. For this reason, it seems clear that process step (a) has a favorable effect on the efficacy of the end-product.
In process step (a) use is preferably made of vapors of concentrated acids or alkalis, and, more specially, concentrated sulfuric acid, acetic acid, formic acid or diethylamine and/or mixtures or peracids with the corresponding concentrated acids, the pulverized substrates or concentrates being acted upon by the vapor of the substance, which is to have an effect on them, in vacuo at room temperature without any further heating, the vapor condensing on the substrate. In German Pat. No. 1,090,821, whose process details may be used in the present invention, the workings of such a process are noted in connection with a partial breaking up of the starting material. The discovery has now been made that on using materials with sterilizing properties, this process gives very high-level effects, per-compounds being more specially preferred in this connection.
Obtaining or producing sterile medicaments from biological tissues, more specially organ tissues, is frequently hard to undertake because, using conventional disinfectants, full sterility may not be produced without great denaturization and loss of the therapeutic efficacy. For this reason, public health authorities have enacted specially tight rules for producing such medicaments, as for example the keeping of isolated groups of animals and breeding them under sterile conditions. Because of such rules or conditions, there has been an overly great increase in price of the products without, however, producing any full and complete warranty for sterility, more specially with respect to viruses. On the other hand, the use as well of antibiotics and/or chemotherapeutic agents having the property of inactivating viruses, is linked with the risk of making the patient allergic.
In experiments with organ powders produced fro virus-infected chicken embryos, it has been possible to make clear that sterility may be produced by a modification of the process for the controlled chemical breaking up of organs for therapeutic purposes (see German Pat. No. 1,090,821). The process is modified in that the substrates, with 5% to 30% of residual water (in comparison with a normal level of 60% to 87% of water in fresh tissues) are acted upon by the vapors of acids in vacuo, there being the addition, to these acid vapors, of the corresponding peracids, more specially of formic acid, acetic acid or sulfuric acid, in a mixing ratio of 1 part of peracid to 1 to 4 parts of the corresponding acid anhydride. Furthermore, vapors of acids may be used, to which, on producing them, a smaller amount of hydrogen peroxide is added than will be necessary for producing the peracid itself, for example there would be the addition to 10 parts of 98% formic acid of 0.1% to 0.8% of 30 % hydrogen peroxide together with 0.5% of concentrated sulfuric acid in place of 10 parts of concentrated formic acid with 1 part of hydrogen peroxide, and in the case of 10 parts of acetic anhydride there would be the addition of only 0.5 to 4 parts of fresh 30% hydrogen peroxide (in place of 5 parts of the same). The useful effect of this process is that the "partial" peracids are less aggressive and may be more readily handled, that is to say with less danger of explosion.
On causing the substrate to be acted upon by vapor, the amount of acid, which becomes condensed on the substrate, is best so controlled that in effect it is equal to a 0.5% to 2% peracid. The condensation operation may, if necessary, be undertaken a number of times by stepping down the vacuum level for a short time before the acid vapors are run into the apparatus again for condensing on the substrate. If the amount of acid is not metered, the sterilization operation may be undertaken for some minutes, whereas if the amounts of acid are limited or measured out for a given amount of substrate, the operation may be undertaken for a number of hours.
The test for sterility of the substrate may be undertaken by ways which are conventional in microbiology and virology by culturing the viruses in hen's eggs or in cell cultures. The testing or assaying of the biological activity of the preparations still in existence is undertaken by bioassay using cell cultures (see V. Paffenholz and K. Theurer: Einfluss von makromolekularen Organsubstanzen auf menschliche Zellen in vitro (The effects of macromolecular organ substances on human cells in vitro), supra) and by animal experiments.
The process is not responsible for any products with a toxic effect, as for example heterocyclic compounds, because the fragments produced do not (because of the absence of water) undergo reaction or conjugation with each other. The peroxides produced at a low level are responsible, on therapeutic application, for a desired stimulating effect in the substrates like that of ozone when therapeutically used. Furthermore, in the case of this process, the acid which has so far not undergone reaction, may be drawn off by stepping up the vacuum.
In other respects, however, it is furthermore possible for neutralization to be caused by blowing in alkali vapors as for example of mercaptoethanol and then drawing off again. By the simultaneous reducing effect, the oxidation effect may, in part, be cancelled out again.
The use of peracid vapors makes necessary the use of acid-resistant plastic piping and glands. The vessel for the peracids may best be connected by means of a three-way cock with the vacuum pump and, or the other hand, with the recipient for the substrate so that, after producing the vacuum in the substrate recipient, the vacuum pump may be cut off and the acid vapors run into the apparatus.
It is furthermore possible for frozen, undried tissue powder to undergo drying before hand (by freeze-drying to get down to the desired residual moisture level, or by drying in the presence of hygroscopic materials such as concentrated sulfuric acid) before the sterilization operation is started. If the residual moisture level in the dried tissues is overly low, they may be processed in a first stage by running water vapor into the apparatus, or by moistening directly.
Details in connection with peracetic acid for "cold sterilization" for thermosensitive instruments have been given in a paper by K. Bansemir, H. Bellinger, K. Disch and W. Kastner in "Hygiene und Medizin" 4 (1979), pages 311 to 316, from which it will be seen that sterilizing effects are produced with a 1% to 2% solution in the case of papova-viruses, Entero-viruses, Poliomyelites type I and furthermore in the case of Coxsackie viruses type B III and Hepatitis-B viruses. An account was furthermore given on toxicological testing. The use of acid solutions as such is, however, not the same as the process of the present invention, because in it the operation takes place at room temperature and with the application in the form of acid vapors, and furthermore on the drying operation the acid, which has not undergone reaction, is drawn off again.
The acid vapor lysis process in vacuo (a) is used as such furthermore for controlled chemical digestion of organs for therapeutic purposes and more specially of fetal, embryonic juvenile tissues, of fetal bovine liver, thymus, spleen, cardiac muscle and brain for producing special preparations for the treatment of neoplasms and diseases of the immune system.
The sort of digestion and degradation of biological tissues is of key importance for their therapeutic effect, as has been seen to be the case for fetal and embryonic organ preparations produced by the process for controlled chemical digestion of organs and for therapeutic purposes (German Pat. No. 1,090,821). The use of such preparations, more specially, of fetal liver, spleen, thymus, cardiac muscle and brain has not been responsible for any incompatibility in the case of animal experiments or in the case of use on humans. The effect of preparations made from fetal and young bovine liver has been surprising in oncology. Although the relation between the carcino-embryonic antigen (CEA) and tumor antigen is known, no report has been come across in the literature to the effect that good prophylactic and therapeutic effects may be produced in the case of experimental tumors by using liver preparations. This has, however, turned out to be the case with preparations produced by the present process in testing on a methyl-cholanthren induced tumor (which was induced some years back in the Sloan Kettering Institute of Cancer Research in New York) on transfer by innoculation to mice.
Furthermore, in animal experiments, it was possible for antibody formation to be forced down to a lower level on using preparations of fetal bovine thymus and fetal spleen. This biological immune suppression is important for the treatment of allergic diseases and has furthermore been seen to take place in the case of man on the therapeutic application of such preparations.
On the other hand, in animal experiments using the hemolysis plaque test, as illustrated in Examples 6 and 7, it was possible to determine that such preparations from fetal cardiac muscle, fetal liver and brain make possible an even more intense stimulation of antibody synthesis than is the case with conventional immune stimulants.
The hemolysis plaque technique makes it possible to get readings for the level of immunological defense from the number of influenced antibody-forming cells in the spleen. If there is a greater plaque formation than in the untreated control, this is a sign for a stepping up of immune defense while, on the other hand, a lower figure for plaque formation indicates a drop in immune response. To this end, mice were sensitized with respect to washed ovine erythrocytes and additionally pre-treated with preparations (produced by the claimed process) from fetal organs, used in an aqueous, diluted form, and then, at different times later on after the injection, the mice were splenectomized. Then a spleen cell suspension was made up, which, together with the ovine erythrocytes and agarose were poured into petri dishes as a thin layer. After incubation for one hour, the development of the plaque-forming cells took place with diluted guinea pig complement, which is necessary for the lysis of the cells in addition. The control group was made up of mice which had not had any organ preparations.
The dry organ powders produced by the present process from fetal and juvenile tissues were taken up directly in physiological sodium chloride and suspended or dilutions in water thereof were prepared. Not only the suspensions, but furthermore the diluted solutions may be injected parenterally.
The process step (b) for the preliminary separating off of ineffective components from the substrate which is to be processed is, on the one hand, for conditioning the affinity sorbent and, on the other hand, for pre-processing the substrate which is to be separated. For this reason, these two preliminary separating operations may be undertaken separately. On the other hand, for separating operation (d) it is possible to make use as well of substrates which have not undergone the part-processes (a) and (b), or have only undergone them in part and which are of different origins. Furthermore, mixtures of different starting materials may be used, although, however, it is best for the ineffective components of the substrate as for example polysaccharides, glycogen, lipids and possible ribonucleic acid (RNA) to be removed conventionally, this furthermore being true for the DNA from the substrate to be separated, if such DNA is not the desired active factor.
The separation off of ineffective or undesired components is not necessary in the case of the substrate to be separated, because the desired active factor is, as we have seen, selectively bound by the affinity sorbent. Only the nucleic acid components (DNA and/or RNA) of the starting substrate or only the protein or peptide fractions of the same are needed for readying the apparatus used for affinity chromatography. For separating, fractional centrifugation of the cell and tissue homogenisates (or suspensions from the dry powder) is used giving a 100,000 g supernatant top layer (see V. Allfrey: Isolation of subcellular components: The Cell: Vol. I, I. Brachet and A. E. Mirsky, Editors, Academic Press, London, 1959--National Cancer Institute Monograph 21: Development of Zonal Centrifuges and Auxillary Systems for Tissue Fractionation and Analysis: National Cancer Institute Bethesda, Md., USA, 1966).
For further isolation, the single fractions of cell nucleic mitochondria, ribosomes and the supernatant top layer are processed with enzymes: for example with ptyalin and maltase and with beta or gamma amylase for the degradation of saccharides, with amyloglucosidase for the degradation of glycogen, with lipase for the degradation of lipids, with RNases for the degradation of ribonucleic acids, with DNase for the degradation of DNA and with proteinases or peptidases (as for example pepsin, papain, trypsin) for the degradation of proteins and peptides. As will be clear, in the preliminary separation of the peptide fractions, no proteinases or peptidases are used and, on producing the nucleic acid fractions, no nucleases are used. (For papers on the isolation of DNA from cells, see: W. Meinke, D. A. Goldstein, M. R. Hall; Anal. Biochem, 58, 82 (1974); G. G. Markov, I. G. Ivanov: Anal. Biochem, 59, 555 (1974); I. G. Ivanov, P. Venkov, G. G. Markov: Preparative Biochem., 5, 219 (1975)).
The degradation products produced by the enzymatic cleaning step may furthermore be removed by dialysis or ultrafiltration to less than 600 molecular weight (see B. L. Williams and K. Wilson: Praktische Biochemie--Grundlagen and Techniken, page 106: publisher: Georg Thieme Verlag, Stuttgart, (1978)). On isolation the ionic environment and the pH of the substrate may only be changed to such a degree that no denaturing of the active substances takes place.
For getting ready for the separation by affinity chromatography in working step (c), the nucleic acid fraction (mostly DNA) or, however, the protein or peptide fraction is coupled to inert carrier material, as far as possible by covalent bonds, to functional chemical groups, without this being responsible for the blocking of reactive bonding sites having a specific effect. A carrier material may, for example, be cellulose, Sephadex hydroxylapatite, polyacryloamide gel, granulated polyethylene, agarose, glass beads, silicone particles, silica gel, zinc oxide, aluminum hydroxide etc. Furthermore, detailed forms of the processes are known in the art (see E. Paoletti et al.: J. Biol. Chem. 249, 3273 (1974)N. Signal et al.: Proc. Nat. Acad. Sci., USA 69, 3537 (1972); R. L. Tsai, H. Green: J. Mol. Biol., 73, 307 (1973); Sutton, D. and Kemp. J. D.: Biochemistry 15, page 3153 (1976); Smith, I. et al.: Anal. Biochem., 48, page 27 (1972); I. A. Lautenberger, S. Linn: J. Biol. Chem. 247, 6176 (1972); and on the question of binding of proteins or peptides to carrier substances see: G. F. B. Schumacher and W. B. Schill: Anal. Biochem. 48, 9 (1972); E. D. Sevier: Anal. Biochem. 74, 592 (1976)). The carrier-bound nucleic acid or protein fraction is now used as a column filling for affinity chromatography (see P. Cuatrecasas: Affinity Chromatography of Macromolecules: Advances in Enzymology. Ed. by A. Meister: 36: 29, 1972. Interscience, New York; F. Friedberg, Chromatography Reviews 14: 121, 1971).
In this respect, a preparative chromatographic column or a combined apparatus for chromatography and electrophoresis on the lines of Free-Flow Electrophoresis may be used (see N. Seiler, I. Thobe, G. Wetner: Elektrophorese im traegerfreien Pufferstrom (electrophoresis in a carrier-free buffer current): Z. Physiol. Chem. 351, 865 (1970); K. Hannig: Z. Anal. Chem. 338, 211 (1964)). The use of carrier-bound filler in the case of this free-flow electrophoresis has so far not been noted in the literature.
The recovery of stimulating fractions from the substrate is undertaken in a separating apparatus with a carrier-bound protein fraction, while the separation of the inhibiting factors takes place in an apparatus with DNA fractions, in the secondary eluate. As soon as the packing of carrier-bound fraction is saturated by absorption of substrate to be separated, the apparatus is rinsed thoroughly with a buffer solution of low ionic strength. This secondary elution takes place of the absorbed fraction. This elution may be undertaken by increasing the ionic strength and changing the pH value of the buffer used. A new process which is specially useful and is very free of undesired effects, makes use of an AC field at a suitable frequency. This sort of elution has so far not been noted in the literature. The useful effect is that the apparatus may be used again, after thorough rinsing with the buffer solution used for primary desorbing. Furthermore, this purpose is furthered by the effect of an electromagnetic field which is normal to the direction of motion of the buffer solution, possibly in combination with electrophoresis. The separation of the substrate in the separating apparatus, readied in process step (c), is best undertaken by sequential or on-off inlet of sample and elution steps. In this respect, in a first step, the components without affinity are produced and then, after cleaning the apparatus by rinsing as a secondary step, elution of the absorbed active factor takes place as detailed earlier. The primary eluate and the secondary eluate have the opposite effective components in them.
For producing tumor-inhibiting factors (e), the separating apparatus is best conditioned with carrier-bound DNA from fetal tissues and/or from different tumor tissues. The absorbing factor may, for this reason, be made up of a single starting substance or of a combination of different substances as for example different sorts of tumors and metastases, such as solid tumors and leukemic cells, which are produced in cell cultures, possibly combined with fetal tissues.
The genus or organ specific factors of the substrates to be separated may be produced or obtained by using carrier-bound factors, which are different for the substrate to be separated. The separating operation takes place using the proteins in question with special degrees of specificity as absorption materials. For this reason, for example, factors, which are contained in the liver, may be isolated from kidney tissue or from an other tissue; on the same lines it is furthermore possible for feto-embryonic and carcino-embryonic antigens and components to be produced from body fluids.
This process is furthermore used for producing inhibiting and stimulating substances with respect to micro-organisms. In this respect, carrier-bound DNA from micro-organisms is used for separating the substrate of the parallel extracts from like or different micro-organisms. In this way it is possible for natural biological antibiotics to be produced while on the other hand stimulating factors take effect on the reproduction and effect of micro-organisms with special functions, as for example of micro-organisms, modified by gene transfer for the synthesis of biological factors as for example insulin and interferon. On these lines, the desired effect of oleophagic micro-organisms may be activated. In the same sort of way, plant factors may be separated, possibly for producing biological plant protection substances or growth factors. On the other hand, the process may furthermore be used for isolating certain gene sections. In this respect, the protein fraction, which has been isolated and identified beforehand, is used in a carrier-bound form for separating off the DNA fraction in question. The DNA gene sections to be isolated will then be in the secondary eluate.
Taking a general view, it may be seen that the present invention is more particularly directed to producing, that is to say obtaining, sterile biological active factors for stimulating, and on the other hand for inhibiting, events in connection with growth, metabolism, synthesis in eukaryoic and furthermore prokaryoic cells and tissues, from cell homogenisates produced from organ tissues, micro-organisms, plant components and/or body fluids (blood, cerebrospinal fluid, amnion-exudates, transsudates, fluids expressed from tissues and urine). A list and further details will now be given to preferred forms of the working steps detailed so far:
(a) the sterilization, with as little undesired effect as possible, of biologically active substances, more specially with respect to viral components and producing better hydrophilic properties, the partial cleavage into molecular sub-units and the addition of radicals from acids and alkalis or bases (used in vapor form), and more specially of concentrated sulfuric acid, acetic acid, formic acid or diethylamine and/or mixtures of peracids with the parallel concentrated acids, in which respect the pulverized substrates or concentrates with the agent for producing the desired effect (and whose boiling point at atmospheric pressure is greater than room temperature) put together in a shut-off or sealed system, but without mixing, are acted upon by such a vacuum as makes possible a change of the agent (for producing the desired effect) into the vapor phase; and, after the desired effect has been produced on the substrate, the unreacted agent is removed again, the starting materials more specially being frozen and pulverized organic tissues, and the chemical agent in vapor form from a preset amount is condensed firstly onto the substrate by lowering the vacuum and, after the desired effect has been produced, the excess agent is removed against by increasing the vacuum and the products so processed are used per se or in the further process steps;
(b) the preliminary removal of ineffective components from the substrate (containing, for example, polysaccharides, lipids, ribonucleic acids and desoxyribonucleic acids (DNA) or, however, protein or peptide fractions) takes place by centrifuging off the cell nuclei and cytoplasmatic components and/or enzymatic degradation, for example using ptyalin and maltase, lipases, RNases, DNases, proteinases or peptidases, dialysis of fragments under a molecular weight of 600 or ultrafiltration;
(c) the coupling of highly purified desoxyribonucleic acid fractions or, on the other hand, of protein or peptide fractions to activated inert carrier material for affinity chromatography of substrates as produced in step (b), in which respect for producing stimulating factors, carrier-bound protein or peptide fractions (or in the case of producing inhibiting factors, carrier-bound DNA fractions) are used for getting ready a preparative chromatographic column or a combined chromatographic and electrophoresis apparatus on the lines of free-flow electrophoresis, if desired, with electrophoretic elution by changing the voltage or current level;
(d) separating the substrate produced in step (b) in the separating apparatus as made ready in step (c), by sample input and elution steps, in which respect, in a first working step the component having no affinity is produced and in a further cleaning rinsing operation undertaken on the apparatus, the elution of the absorbing active factor is undertaken by using an electric DC field or an AC field of the necessary frequency and/or of an electromagnetic-field or by changing the buffer solution;
(e) producing tumor-inhibiting factors by affinity chromatography of substrates, from fetal and juvenile normal tissues and/or body fluids from healthy individuals, or carrier-bound desoxyribonucleic acid from fetal tissues and/or from different tumor tissues or from tumor cell lines cultivated in vitro by themselves or in combination by secondary elution of carrier-bound fractions (whereas the stimulating factors are produced by undergoing primary elution);
(f) producing stimulating factors as well from substrates produced from normal tissues and/or body fluids of healthy individuals by affinity chromatography on carrier-bound protein or peptide fractions from healthy tissues by a second elution operation, while the inhibiting fractions are in the first eluate;
(g) producing organ specific and/or fetal components using, on the same general lines, the DNA, RNA, or protein (or peptide) fractions of the parallel organ tissue, more specially from fetal, embryonic and/or juvenile tissues of healthy individuals, as for example liver, thymus, spleen, cardiac muscle, brain etc.; and
(h) producing inhibiting or stimulating substances for micro-organisms or plants from the parallel extracts making use of carrier-bound nucleic acids.
The isolated factors may be used in aqueous solutions or incorporated in liposomes or in a water-in-oil emulsion (see German Pat. Nos. 2,650,502, 2,656,333 and 2,640,707) in an aqueous phase and, on the other hand, after further separating and cleaning their structures may be elucidated and partly or completely synthesized, this being a further part of the present invention. The isolated, inhibiting proteins or peptides may furthermore be effective for the isolation of certain desoxyribonucleic acids (which may be used as gene sections) in a carrier-bound form for affinity-chromatographic separation of such DNA mixtures or solutions, the DNA sections, which are to be enriched or isolated, being present in the secondary eluate.